十字花科根瘤病生物防治資源之開發與應用

Abstract

十字花科根瘤病（clubroot disease of Brassicaceae）是由絕對寄生之內部寄生性黏菌 Plasmodiophora brassicae Woronin 所引起之十字花科重要病害。目前本病害多以施用石灰提高土壤酸鹼值，進行防治，但也造成土壤鹽鹼化之問題，因此開發其他防治方法，實為刻不容緩之事。本論文目標有三，一為藉由實際的田間調查，了解臺灣根瘤病之發病狀況，以及農民耕作習慣與防治施行等；二為嘗試探討臺灣 P. brassicae 小種的不同，提供十字花科作物育種及田間品系種植選擇之參考資訊；三為開發生物防治資源，藉由分離田間土壤中具芥子酶（myrosinase）活性之微生物，分解硫配醣體（glucosinolate）產生異硫氰化物（isothiocyanate），進行十字花科根瘤病生物防治燻蒸測試。針對上述目標，實際調查田間發病狀況，全省各主要高冷蔬菜產區皆有此病害之發生，發病率於10至100%不等。在根瘤病菌小種分群方面，我們自臺灣8縣市23處病田採得樣本，嘗試進行分子分群試驗，結果發現臺灣根瘤病菌無法以 rDNA 進行分群，但以一特殊序列分子標記可發現臺灣平地和高山的菌株分屬不同的小種，可作為未來進一步分群之用。在分離具芥子酶活性之微生物方面，自臺灣許多十字花科蔬菜種植田，分得19株具有芥子酶活性的真菌，並進行其中5株真菌之生長特性及防治測試，發現由臺北市北投區分離之Aspergillus japonicus var. aculeatus，不論在中溫（20-30°C）生長速度，及於植物根部根圈定棲能力，皆有不錯之能力，而於溫室和田間試驗中，當該菌和菜籽粕共同施用於土壤時，對根瘤病皆得到穩定且優良的防治效果。藉由本論文的研究，找到頗具生物防治潛力之真菌，期待未來能結合耕作防治，應用於十字花科根瘤病之防治，提供有效且對環境無不良影響的防治新方法。

Clubroot disease of Brassicaceae, caused by an obligate endo-parasitic slime mold Plasmodiophora brassicae Woronin, is one of serious problems of cruciferous crop. Currently, one of major control methods for clubroot disease is to elevate pH value by adding lime into soil; however, detrimental effects including soil alkalization and hardening often occur and exhibit impact on plant growth and environment. Thus, alternative and effective control measures are currently in need. The goal of our study is to explore bio-control resources and develop new disease control method for clubroot disease. To achieve this goal, three specific aims include: (i) to investigate the occurrence of clubroot disease in the high elevation fields of Taiwan; (ii) to collect and differentiate P. brassicae races; (iii) to isolate myrosinase-producing microbes and develop a biofumigation control protocol based on degradation products, isothiocyanate, of glucosinolates. Our field survey revealed that clubroot disease occurs in the fields of high elevation growth areas with incidence of 10-100%. Twenty three clubroot samples collected from 8 different counties in Taiwan were subjected to race differentiation experiments. We failed to differentiate these samples by rDNA sequence, but samples from plain fields or high mountains can be separated into 2 pathotypes based on specific sequence marker. Furthermore, 19 myrosinase-producing fungal isolates were isolated from field samples. Among these fungi, 5 isolates were subjected to growth and disease control assays and one isolate from Beitou, Aspergillus japonicus var. aculeatus showed good growth ability at 20-30°C and extensive root colonization capability. In both green house and field tests, this isolate also exhibited good disease control results when combining with canola meal. In this study, a myrosinase-producing fungal isolate was identified and combination of this bio-control agent with other culture control measures may potentially provide an effective and environmental-friendly method to control the clubroot disease.